Neurodegeneration in frontotemporal lobar dementia with ubiquitinated inclusions (FTLD-U), amyotrophic lateral sclerosis (ALS), and other delayed-onset diseases associated with degeneration of aging neurons involves a deficit in the degradation of the TAR DNA binding protein (TDP-43). This deficit is apparent through the characteristic pathology of a cross-section of neurodegenerative diseases, i.e., accumulation and aggregation of protein inclusions positive for TDP-43 and ubiquitin (Ub) in ALS, FTLD-U, and a large fraction of Alzheimer's Disease (AD) and Parkinson's Disease (PD) cases. Ub is a small protein conjugated singly or in polymeric chains to substrate proteins like TDP-43 that has the capacity to alter substrate fate, e.g. degradation, (in) activation, or relocation out of specific cellular compartments or protein complexes. The proposed research identifies the determinants of TDP-43 degradation, and determines the role of K63-linked polyubiquitin in rerouting of TDP-43 from degradation pathways to disease-characteristic aggregates. Half-life studies in cortical neurons and glia and in human cell lines measure the contributions of cellular alternatives for protein degradation, the proteasome and lysosome, to the degradation kinetics of endogenously expressed TDP-43. In addition, K63-linked Ub chains on TDP-43 and their association with the cell's quality control and aggregation-control machinery are identified by experiments in a cell line engineered to express modified Ub. Components of cellular quality control machinery have been shown to engage TDP-43 in neurodegenerative tissue and in models of neurodegeneration, particularly those which test the effects of insufficient proteasome or lysosome activity on substrates such as TDP-43. This insufficiency may passively develop in neurons during the course of aging or may develop due to environmental factors that affect sporadic neurodegeneration. Significant quantitative differences in interactions between TDP-43 and the Ub-dependent proteasome, lysosome, and quality control systems of cells in disease tissue will be identified. Interactions relevant to disease progression (biomarkers) or etiology (therapeutic targets) are to be confirmed by immunohistochemical staining in postmortem tissue from FTLD-U and ALS neurodegenerative disease. Finally, biochemical assays will be employed to identify specific therapeutic target(s) that deposit K63-linked Ub chains onto TDP-43; K63 Ub linkages significantly increase in neurodegenerative disease and published models of TDP-43 proteinopathy. ALS-specific mutations of TDP-43 will be tested for changes in the propensity of the protein to interact with identified interaction partners, particularly the Ub ligase that deposits K63-linked Ub chains onto TDP-43. The proposed measurements of alternative Ub chains and identification of novel disease-related proteins will rely on mass spectrometry methods and software developed by the PI and his mentors at the Emory proteomics facility and Center for Neurodegenerative Disease, where the proposed research takes place.